Many viruses and toxins require inactivation before they can be used as vaccines and chemical modifying agents such as formaldehyde have been widely used in vaccine production. Notable examples include, for instance, polio, tetanus, diphtheria, botulinum and anthrax vaccines. Protein modification by formaldehyde is complex and involves the chemical modification of several amino acid residues (such as arginine and lysine) and also the formation of cross-links (methylene bridges) which can lead to extensive protein aggregation (Metz et al. (2004) J. Biol Chem., 279: 6235-6243).
The use of formaldehyde for vaccine production does have its drawbacks, the most common of which is the modification of protein structure which results in a loss of immunogenic epitopes and an over all decrease in the immunogenicity of the protein (Vani et al. (2006) J. Immunol Methods. 317, 80-89). However, for some small proteins, formaldehyde has been reported to enhance the immune response through protein aggregation. For instance, it has been reported that formaldehyde treatment increases the immunogenicity and decreases the toxicity of low molecular weight Haemophilus ducreyi cytolethal distending toxins (HdCDT) (Lagergard et al. (2007) Vaccine: 25, 3606-14). Problems associated with formaldehyde treatment, particularly the frequent reduction of immunogenicity and the complexity of the final product through aggregation, have led to a decline in its use, especially with the introduction of recombinant technology which provides a means of rendering harmful proteins inactive through site directed mutagenesis.
The botulinum neurotoxins (BoNTs) are a family of seven antigenically different protein toxins (serotypes A-G). These neurotoxins are extremely potent neuroparalytic agents which act primarily at the peripheral nervous system where they inhibit the release of acetylcholine at the neuromuscular junction (Niemann (1991) In Sourcebook of Bacterial Protein Toxins (Alouf, J. E. & Freer, J. H. eds.), pp. 303-348, Academic Press, London). This is mediated via highly specific zinc-dependent endopeptidase activity directed at small proteins involved in the fusion and release of synaptic vesicles. The botulinum neurotoxins are structurally similar; they have 30-40% sequence homology and, as diagrammatically shown immediately below, each neurotoxin consists of a heavy chain (100 kDa) and a light chain (50 kDa) linked by a disulphide bridge (Niemann, 1991, as above). Despite structural similarities, antisera raised against purified neurotoxins show no cross-protection between the neurotoxin serotypes and thus necessitate the development of a separate vaccine for each serotype. In addition, within each serotype, various subtypes exist (Minton (1995) In: Current Topics in Microbiology and Immunology 195 ‘Clostridial Neurotoxins’ (Montecucco, C., ed.) pp. 161-194, Springer, Berlin). Since these subtypes differ in their antigenic properties, the presence of these toxin variants needs to be taken into account in vaccine design to ensure adequate cross protection.
Structure of Botulinum Neurotoxins and the LHN Fragment

The C-terminal 50 kDa fragment (HC fragment) is responsible for receptor-binding at the presynaptic nerve surface (Halpern & Loftus (1993) J. Biol. Chem. 268, 11188-11192); (Shone et al. (1985) Eur. J. Biochem., 151, 75-82). The N-terminal 50 kDa portion of the heavy chain (HN fragment) is involved in translocation of the enzymatically active light chain to within the nerve terminal (Shone et al. (1987) Eur. J. Biochem., 167, 175-180). Removal of the HC domain from the BoNT leaves a fragment (LHN) consisting of the light chain and translocation domain which, although virtually non-toxic, is stable and soluble. Any residual toxicity is eliminated by double mutations in the enzymatic domain yielding a non-toxic LHN vaccine.
Tetanus and the botulinum neurotoxin are extremely potent, bacterial neurotoxins produced by various strains of Clostridia. The botulinum neurotoxins consist of seven distinct serotypes and a separate vaccine is required for each. First generation tetanus and botulinum vaccines consist of purified or partially purified toxins treated with formaldehyde to eliminate the neurotoxic action of these protein toxins. In the case of the botulinum toxins, complete detoxification requires incubation of toxin preparations for over three weeks in order to generate the toxoid vaccine derivative. In addition, since the botulinum toxins are in the form of high molecular weight (300-900 kDa) protein complexes, the resulting toxoid product is an extremely heterogeneous mixture consisting of very high molecular weight species (Singh et al. (1989) Toxicon 27, 403-410). Another disadvantage of formaldehyde treatment is that in the case of some of the botulinum toxoid serotypes (e.g., type A), several epitopes are destroyed in the prolonged toxoiding process (Hallis et al. (1993) Characterization of monoclonal antibodies to BoNT/A. In: Botulinum and Tetanus Neurotoxins, (DasGupta, B., Ed.) p 433-436, Plenum Press).
Second generation botulinum vaccines are based on non-toxic fragments of the botulinum toxins and are designed to eliminate the requirement for a detoxification step with formaldehyde. One such vaccine candidate is the LHN fragment (light chain domain plus HN translocation domain), which consists of the N-terminal two-thirds of the botulinum neurotoxin moiety. This fragment is a single chain polypeptide which lacks the ability of the parent neurotoxin to bind to nerve endings and in addition may contain one or more amino acid mutations within the light chain domain to render it completely non-toxic. In addition to being non-toxic, the LHN fragments are easy to characterize being monomeric in solution with none of the complex aggregation associated with the corresponding toxoid which is normally purified as a toxin complex. See, for instance, U.S. patent application Ser. Nos. 11/717,713 and 11/077,550, which are herein incorporated by reference in their entireties.
The diversity within the BoNT family is a major problem for vaccine design and the extent of this problem is only now becoming appreciated. While it is widely recognised that the different BoNT serotypes are antigenically distinct and require separate vaccines, it is less well appreciated that antigenically different sub-types exist within each of the main BoNT serotypes (Smith et al (2005) Infect Immum, 73:5450-5457). BoNT/A, for example, is now known to contain at least 4 sub-types and a similar number of sub-types exists within the BoNT/B family. Differences in the primary structure within the various subtypes are reflected in differences in their antigenic profile, with the result that a vaccine which protects against one toxin subtype may not protect against another. Providing adequate protection against toxin sub-types is an important consideration for vaccine design.
There is therefore a need for improved botulinum and tetanus vaccines, such as vaccines having one or more of: an improved protective effect; improved stability; improved cross-serotype protection; and improved cross-subserotype protection.
In addition to the above-mentioned clostridial species, Clostridium difficile is now a major problem as a healthcare acquired infection (HCAI). The bacterium causes nosocomial, antibiotic-associated diarrhoea and pseudomembranous colitis in patients treated with broad-spectrum antibiotics. Elderly patients are most at risk from these potentially life-threatening diseases and incidents of hospital infection have increased dramatically over the last 10 years. Strains of C. difficile produce a variety of virulence factors, notable among which are several protein toxins: Toxin A, Toxin B and, in some strains, a binary toxin which is similar to Clostridium perfringens lota toxin. Toxin A is a large protein cytotoxin/enterotoxin, which plays a key role in the pathology of infection and which also appears to have some role in the gut colonisation process. Toxin B, which is primarily a cytotoxin, appears to act synergistically with Toxin A.
Antibodies to Toxins A and B have been shown to protect against Clostridium difficile associated disease and hence non-toxic fragments of either Toxin A, B, or the binary toxin have potential as vaccines or as antigens for producing therapeutic antibodies. Recombinant fragments of Clostridium difficile toxins, however, generally do not produce a strong neutralising response in animals in conjunction with an adjuvant such as aluminium hydroxide (e.g., Alhydrogel).
Again, there is therefore the need for an improved C. difficile vaccine such as a vaccine having one or more of: an improved protective effect; and improved stability.
Anthrax is an acute infectious disease in humans and animals that is caused by the bacterium Bacillus anthracis and which in some forms is lethal. Protective antigen (PA), lethal factor (LF) and edema factor (EF) are components of anthrax toxin which play a key role in mediating its biological effects and the disease. PA contains domains that bind cell receptors and which can effect the translocation of EF and LF into cells. Once inside the cell, LF and EF have lytic actions via different mechanisms. PA, EF, and LF on their own are non-toxic and are only active in combinations in which one component is PA.
Since PA is the common factor required for both the actions of LF and EF, a recombinant fragment has been assessed as a vaccine for anthrax. Recombinant PA, however, does not elicit a strong protective response against the disease and there have also been issues with its stability.
There is therefore the need for an improved anthrax vaccine, such as a vaccine having one or more of: an improved protective effect; and improved stability.